scholarly journals Gating currents associated with Na channels in canine cardiac Purkinje cells.

1990 ◽  
Vol 95 (3) ◽  
pp. 439-457 ◽  
Author(s):  
D A Hanck ◽  
M F Sheets ◽  
H A Fozzard
Keyword(s):  
Purkinje Cells ◽  
Single Channel ◽  
State Transitions ◽  
Na Channel ◽  
Channel Measurements ◽  
Na Channels ◽  
Gating Currents ◽  
Closed State ◽  
Conductance Curve ◽  
Cardiac Purkinje Cells

Gating currents (Ig) were recorded in single canine cardiac Purkinje cells at 10-12 degrees C. Ig characteristics corresponded closely to macroscopic INa characteristics and appeared to exhibit little contamination from other voltage-gated channels. Charge density predicted by peak INa was 0.14-0.22 fC micron -2 and this compared well with the measured value of 0.19 +/- 0.10 fC micron -2 (SD; n = 28). The charge-voltage relationship rose over a voltage similar to the peak INa conductance curve. The midpoints of the two relationships were not significantly different although the conductance curve was 1.5 +/- 0.3 (SD; n = 9) times steeper. Consistent with this observation, which predicted that a large amount of the gating charge would be associated with transitions close to the open state, an analysis of activation from Hodgkin-Huxley fits to the macroscopic currents showed that tau m corresponded well with a prominent component of Ig. Ig relaxations fitted two exponentials better than one over the range of voltages in which Na channels were activated. When the holding potential was hyperpolarized, relaxation of Ig during step depolarizations to 0 mV was prolonged but there was no substantial increase in charge, further suggesting that early closed-state transitions are less in charge, further suggesting that early closed-state transitions are less voltage dependent. The single cardiac Purkinje cell appears to be a good candidate for combining Ig and single-channel measurements to obtain a kinetic description of the cardiac Na channel.

scholarly journals Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin.

1995 ◽  
Vol 106 (4) ◽  
pp. 617-640 ◽  
Author(s):  
M F Sheets ◽  
D A Hanck
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Na Channel ◽  
Total Charge ◽  
Na Channels ◽  
Gating Currents ◽  
Gating Charge ◽  
Cardiac Purkinje Cells ◽  
Voltage Dependent ◽  
Cardiac Sodium Channels

The gating charge and voltage dependence of the open state to the inactivated state (O-->I) transition was measured for the voltage-dependent mammalian cardiac Na channel. Using the site 3 toxin, Anthopleurin-A (Ap-A), which selectively modifies the O-->I transition (see Hanck, D. A., and M. F. Sheets. 1995. Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Qmax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-->I. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O-->I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O-->I transition determined. The O-->I transition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O-->I transition at 0 mV could also be estimated (0.53 ms, approximately 12 degrees C). Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O-->C and O-->I rate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.

scholarly journals Kinetic effects of quaternary lidocaine block of cardiac sodium channels: a gating current study.

1994 ◽  
Vol 103 (1) ◽  
pp. 19-43 ◽  
Author(s):  
D A Hanck ◽  
J C Makielski ◽  
M F Sheets
Keyword(s):  
Antiarrhythmic Drugs ◽  
Na Channel ◽  
Na Channels ◽  
Gating Currents ◽  
Cardiac Purkinje Cells ◽  
Affinity Receptor ◽  
New Interpretation ◽  
Kinetic Effects ◽  
Cardiac Sodium Channels ◽  
Modulated Receptor

The interaction of antiarrhythmic drugs with ion channels is often described within the context of the modulated receptor hypothesis, which explains the action of drugs by proposing that the binding site has a variable affinity for drugs, depending upon whether the channel is closed, open, or inactivated. Lack of direct evidence for altered gating of cardiac Na channels allowed for the suggestion of an alternative model for drug interaction with cardiac channels, which postulated a fixed affinity receptor with access limited by the conformation of the channel (guarded receptor hypothesis). We report measurement of the gating currents of Na channels in canine cardiac Purkinje cells in the absence and presence of QX-222, a quaternary derivative of lidocaine, applied intracellularly, and benzocaine, a neutral local anesthetic. These data demonstrate that the cardiac Na channel behaves as a modulated rather than a guarded receptor in that drug-bound channels gate with altered kinetics. In addition, the results suggest a new interpretation of the modulated receptor hypothesis whereby drug occupancy reduces the overall voltage-dependence of gating, preventing full movement of the voltage sensor.

Is there a second external lidocaine binding site on mammalian cardiac cells?

1989 ◽  
Vol 257 (1) ◽  
pp. H79-H84 ◽  
Author(s):  
L. A. Alpert ◽  
H. A. Fozzard ◽  
D. A. Hanck ◽  
J. C. Makielski
Keyword(s):  
Binding Site ◽  
Cardiac Arrhythmias ◽  
Local Anesthetics ◽  
Sodium Current ◽  
Cardiac Cells ◽  
Na Channel ◽  
Functional Difference ◽  
Na Channels ◽  
Internal Perfusion ◽  
Cardiac Purkinje Cells

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

Brefeldin A inhibition of apical Na+ channels in epithelia

1996 ◽  
Vol 270 (1) ◽  
pp. C138-C147 ◽  
Author(s):  
R. S. Fisher ◽  
F. G. Grillo ◽  
S. Sariban-Sohraby
Keyword(s):  
Cultured Cells ◽  
Single Channel ◽  
Noise Analysis ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Na Channel ◽  
Na Channels ◽  
Na Transport ◽  
Channel Current ◽  
Vacuolar System

Brefeldin A (BFA) is used to probe trafficking of proteins through the central vacuolar system (CVS) in a variety of cells. Transepithelial Na+ transport by high-resistance epithelia, such as A6 cultured cells, is inhibited by BFA. Apical Na+ channels, as well as basolateral pumps and K+ channels, are complex proteins that probably traverse the CVS for routing to the plasma membrane. BFA (5 micrograms/ml) decreases transepithelial Na+ current near zero and increases resistance reversibly after 4 h. Longer exposures are toxic. When tissues were treated for 20 h with 0.2 microgram/ml BFA, Na+ transport also was reversibly inhibited. Using noise analysis, we found that BFA drastically reduced apical Na+ channel density. The increase in single channel current was consistent with cell hyperpolarization. After apical permeabilization with nystatin, changes in transepithelial current reflect changes in basolateral membrane transport. Transport at this membrane was inhibited by ouabain and cycloheximide, but not by BFA. After BFA, aldosterone was ineffective, suggesting that an intact CVS is required for stimulation by this hormone. Thus BFA inhibition of Na+ transport is localized at the apical membrane. Implications for channel turnover as a mechanism for regulating the Na+ transport rate are discussed.

Cholinergic modulation of apical Na+ channels in turtle colon: analysis of CDPC-induced fluctuations

1990 ◽  
Vol 259 (4) ◽  
pp. C668-C674 ◽  
Author(s):  
D. J. Wilkinson ◽  
D. C. Dawson
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Rate Coefficients ◽  
Corner Frequency ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Na Channels ◽  
Na Current

Current fluctuation analysis was used to investigate the properties of apical Na+ channels during muscarinic inhibition of active Na+ absorption. A reversible Na+ channel blocker, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), was used to induce fluctuations in the short-circuit current (I(sc)). Power density spectra of the CDPC-induced fluctuations exhibited a clearly discernible Lorentzian component, characterized by a corner frequency that was linearly related to CDPC concentration between 20 and 100 microM. The on (k'on) and off (k(off)) rate coefficients for the CDPC blocking reaction were k'on = 11.1 +/- 0.8 rad.s-1.microM-1 and k(off) = 744 +/- 53 rad/s, and the microscopic inhibition constant was 67 microM (n = 11). CDPC blocking kinetics were not significantly different after inhibition of Isc by 5 microM serosal carbachol. Single-channel Na+ current (iNa) and the density of open and blocked Na+ channels (N(ob)) were estimated from the fluctuations induced by 40 microM CDPC. Under control conditions, iNa was 0.43 +/- 0.05 pA and N(ob) was 251 +/- 42 X 10(6)/cm2 (n = 10). After exposure to serosal carbachol (2-10 microM) for 60 min, Na+ current and N(ob) were reduced by approximately 50%, but iNa was not changed significantly. These results indicate that muscarinic inhibition of electrogenic Na+ absorption was associated with a reduction in the number of open Na+ channels in the apical membrane. They also suggest that this downregulation of transport involved a coordinated decrease in both apical and basolateral membrane conductances.

scholarly journals Sodium channel subconductance levels measured with a new variance-mean analysis.

1988 ◽  
Vol 92 (4) ◽  
pp. 413-430 ◽  
Author(s):  
J B Patlak
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Na Channel ◽  
Na Channels ◽  
Open Probability ◽  
Open Time ◽  
Flexor Digitorum Brevis ◽  
Dissociated Cells ◽  
A New Technique ◽  
Flexor Digitorum

The currents through single Na+ channels were recorded from dissociated cells of the flexor digitorum brevis muscle of the mouse. At 15 degrees C the prolonged bursts of Na+ channel openings produced by application of the drug DPI 201-106 had brief sojourns to subconductance levels. The subconductance events were relatively rare and brief, but could be identified using a new technique that sorts amplitude estimates based on their variance. The resulting "levels histogram" had a resolution of the conductance levels during channel activity that was superior to that of standard amplitude histograms. Cooling the preparation to 0 degrees C prolonged the subconductance events, and permitted further quantitative analysis of their amplitudes, as well as clear observations of single-channel subconductance events from untreated Na+ channels. In all cases the results were similar: a subconductance level, with an amplitude of roughly 35% of the fully open conductance and similar reversal potential, was present in both drug-treated and normal Na+ channels. Drug-treated channels spent approximately 3-6% of their total open time in the subconductance state over a range of potentials that caused the open probability to vary between 0.1 and 0.9. The summed levels histograms from many channels had a distinctive form, with broader, asymmetrical open and substate distributions compared with those of the closed state. Individual subconductance events to levels other than the most common 35% were also observed. I conclude that subconductance events are a normal subset of the open state of Na+ channels, whether or not they are drug treated. The subconductance events may represent a conformational alteration of the channel that occurs when it conducts ions.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6)

1992 ◽  
Vol 263 (3) ◽  
pp. F392-F400 ◽  
Author(s):  
Y. Marunaka ◽  
N. Hagiwara ◽  
H. Tohda
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Channels ◽  
Distal Nephron ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Cardiac sodium channels expressed in a peripheral neurotumor-derived cell line, RT4-B8

1996 ◽  
Vol 270 (5) ◽  
pp. C1522-C1531 ◽  
Author(s):  
D. Zeng ◽  
J. W. Kyle ◽  
R. L. Martin ◽  
K. S. Ambler ◽  
D. A. Hanck
Keyword(s):  
Cell Line ◽  
Northern Blot ◽  
Single Channel ◽  
Small Time ◽  
Na Channel ◽  
Mrna Levels ◽  
Na Channels ◽  
Subunit Mrna ◽  
Cardiac Phenotype ◽  
Cardiac Sodium Channels

RT4-B is one of several cell lines derived from a multipotent stem cell line, RT4-AC, which originated from a rat peripheral neurotumor. Based on Northern blot and ribonuclease protection experiments, RT4-B8 cells have been proposed to express rat cardiac Na channel mRNA as the major isoform. We report here direct electrophysiological evidence that the expressed voltage-gated Na channels in the RT4-B8 cell line are of the cardiac phenotype with no evidence for subpopulations expressing other Na channel isoforms. Current activation half point (conductance) was -41 +/- 5 mV (n = 7) and the steady-state voltage-dependent availability half point was -89 +/- 1 mV. As expected for cardiac Na channels, the half concentration of block for tetrodotoxin block was 0.74 microM, for saxitoxin (STX) was 0.15 microM, and for the class 2B divalent cation Cd2+ was 67 microM. Block was well described by single-site dose-response relationships with no indication of a subpopulation with "neuronal" affinity. Single-channel conductance (140 mM Na+) was 10 pS and predicted the average number of channels open at peak Na current to be 3 channels/microns2. [3H]STX binding data were also consistent with a single population of low-affinity STX binding sites and predicted channel density to be 11 sites/microns2. No inwardly or outwardly rectifying K or Ca currents were detected electrophysiologically, although in some cells a small time-independent Cl current was detected. Reverse transcription-polymerase chain reaction of mRNA isolated from RT4-B8 cells demonstrated the presence of rat cardiac (rH1) and brain IIa alpha-subunit mRNA, as well as mRNA for the Na channel beta 1-subunit. Northern blot analysis confirmed the predominance of the rat cardiac Na mRNA compared with brain IIa. The beta 1-subunit mRNA levels were significantly lower than those detected in rat brain and heart mRNA but were comparable to the low level of beta 1-subunit mRNA detected in isolated rat ventricular myocytes.

scholarly journals Gating of Na channels. Inactivation modifiers discriminate among models.

1987 ◽  
Vol 89 (2) ◽  
pp. 253-274 ◽  
Author(s):  
T Gonoi ◽  
B Hille
Keyword(s):  
Proteolytic Enzymes ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Cell Voltage ◽  
Na Channel ◽  
Na Channels ◽  
Negative Direction ◽  
Conductance Curve ◽  
Voltage Curve ◽  
Na Currents

Macroscopic Na currents were recorded from N18 neuroblastoma cells by the whole-cell voltage-clamp technique. Inactivation of the Na currents was removed by intracellular application of proteolytic enzymes, trypsin, alpha-chymotrypsin, papain, or ficin, or bath application of N-bromoacetamide. Unlike what has been reported in squid giant axons and frog skeletal muscle fibers, these treatments often increased Na currents at all test pulse potentials. In addition, removal of inactivation gating shifted the midpoint of the peak Na conductance-voltage curve in the negative direction by 26 mV on average and greatly prolonged the rising phase of Na currents for small depolarizations. Polypeptide toxins from Leiurus quinquestriatus scorpion and Goniopora coral, which slow inactivation in adult nerve and muscle cells, also increase the peak Na conductance and shift the peak conductance curve in the negative direction by 7-10 mV in neuroblastoma cells. Control experiments argue against ascribing the shifts to series resistance artifacts or to spontaneous changes of the voltage dependence of Na channel kinetics. The negative shift of the peak conductance curve, the increase of peak Na currents, and the prolongation of the rise at small depolarization after removal of inactivation are consistent with gating kinetic models for neuroblastoma cell Na channels, where inactivation follows nearly irreversible activation with a relatively high, voltage-independent rate constant and Na channels open only once in a depolarization. As the same kind of experiment does not give apparent shifting of activation and prolongation of the rising phase of Na currents in adult axon and muscle membranes, the Na channels of these other membranes probably open more than once in a depolarization.

Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells

2007 ◽  
Vol 293 (5) ◽  
pp. F1666-F1677 ◽  
Author(s):  
Ling Yu ◽  
Hui-Fang Bao ◽  
Julie L. Self ◽  
Douglas C. Eaton ◽  
My N. Helms
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Single Channel ◽  
Na Channel ◽  
Dependent Manner ◽  
Channel Measurements ◽  
Distal Nephron ◽  
Open Probability ◽  
A6 Cells ◽  
A Cell

Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O2−) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 μM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O2−(with the O2−scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 ± 0.03 to 0.03 ± 0.01. We also found that increasing endogenous O2−in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 ± 0.05) after superoxide dismutase and 1.5 μM NO coincubation (0.21 ± 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O2−by ∼30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2−alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.

Sign in / Sign up

Export Citation Format

Share Document